Whether apoptotic stimuli arise from the nucleus, cell membrane surface, or the mitochondria, ultimately, the stimuli converge on a process of activation of a family of cysteine proteases known as the caspases (cysteine aspartases). Activation of caspase family members mediate programmed cell death in normal physiology and a number of diseases, with caspase-3 standing at the center of the cell death program. Existing in a non-active pro-enzyme form in the cytosol of resting cells, caspase-3 is one key "effector" protease when activated. Thus, to monitor the final commitment of cells to death pathways, the need exists to directly quantify the enzymatic activity of caspase-3 in vivo. To meet this challenge, we designed and synthesized a new class of peptide-based imaging agents that can penetrate the cell membrane into the cytosolic compartment. These cell-penetrating optical imaging agents contain quenched fluorophores flanking target protease sequences which are cleaved and activated by caspase-3. Upon cleavage, these agents show caspase-3-dependent fluorescence signal amplification, thereby enabling high quality enzyme-specific molecular imaging of intracellular processes in vivo. Importantly, during our last funding cycle Activation of caspase family members mediate programmed cell death in normal physiology and a number of diseases, with caspase-3 standing at the center of the cell death program. RGCs are particularly accessible through an intravitreal approach, a routine ophthalmological procedure performed everyday in the clinic. Thus, we plan to exploit this unanticipated and exciting cell-type-specific property of our peptides in retinal pathology for molecular imaging applications, combined diagnostic-therapeutic ("theragnostic") anti-apoptotic applications as well as mechanism-based studies. In this way, we will generate agents for focal retention of fluorescence, an optical "hot spot", in target cells corresponding to enzymatically active caspase-3 in vivo as well as real-time imaging of therapeutic delivery to single target cells. These activities benefit from the combination expertise of this applicant team in chemistry, molecular imaging, biochemistry, and vision biology. PUBLIC HEALTH RELEVANCE: Activation of enzymes that mediate programmed cell death occurs in normal physiology and in a number of diseases. One these enzymes, known as caspase-3, stands at the center of the cell death program. Thus, to monitor the final commitment of cells to death pathways, the need exists to directly quantify the enzymatic activity of caspase-3 in vivo. To meet this challenge, we have designed and synthesized a new class of peptide-based imaging agents that can silently penetrate the cell membrane into interior cellular compartments and be activated specifically by caspase-3 to emit visible fluorescence. Thus, we plan to exploit this property of our peptides in retinal pathology for molecular imaging applications, and combined diagnostic-therapeutic ("theragnostic") anti-apoptotic applications. These activities benefit from the combination expertise of this applicant team in chemistry, molecular imaging, biochemistry, and vision biology.